The long-term goal of this project is to define how thrombin triggers cellular responses and the roles of thrombin signaling in biology and disease. Thrombin's actions on platelets are critical for homeostasis and thrombosis, and its actions on leukocytes, endothelial, and mesenchymal cells may contribute to inflammatory and proliferative processes. Thrombin activates platelets and other cells at least in part via G protein- coupled protease-activated receptors (PARs). Further study of PARs promises to reveal new modes of receptor activation, interaction, and intracellular sorting. Defining the roles of PARs in vivo may provide new strategies for the prevention and treatment of thrombosis and other diseases. The following questions will be asked: 1) Does mPAR3 mediate transmembrane signaling on its own or, instead, act as a cofactor for mPAR4 activation? The paradigm of one receptor's binding a ligand that in turn activates a distinct receptor is unprecedented for GPCRs. PAR3 and PAR4 are the thrombin receptors known to participate in mouse platelet activation. Knockout for PAR3 attenuated activation of mouse platelets by low concentrations of thrombin, but expression of mPAR3 in heterologous systems has not conferred signaling. We hypothesize that mPAR3 functions by binding thrombin and promoting cleavage and activation of Mpar4. 2) Do cofactor mechanisms play a more general role in PAR activation? Results from Aim 1 will likely cast mPAR3 as a cofactor for ability of known protease cofactors to promote PAR activation will be assessed. 3) Do hPAR1 and Hpar4) have distinct roles and/or interact in human platelets? In contrast to mouse platelets, human platelets use PAR1 and PAR4 to mediate thrombin signaling, and activation of either receptor appears to be sufficient to trigger robust responses. Are hPAR1 and hPAR4 redundant or do they interact or serve at least partially distinct functions? Ability to activate distinct downstream signaling pathways and/or mediate signaling to proteases other than thrombin will be assessed. 4) How is activated PAR1 internalized and delivered to lysosomes? Internalization and sorting of activated receptors to lysosomes is important for the long-term down- regulation of signaling pathways. In multiple cell types, sorting of activated PAR1 to lysosomes is important for the long-term down- regulation of signaling pathways. In multiple cell types, sorting of activated PAR1 to lysosomes is rapid and robust. Thus PAR1 is a useful model system for the study of receptor down-regulation in mammalian cells. We will use complementation cloning to identify new molecules involved in PAR1 trafficking.